During the first 10 years of this application, our focus was on the role of the dopaminergic system in Rett syndrome (RS). Over the last 5 years, the focus has also been on volumetric measurements of various Rett syndrome brain structure as a function of age. As more interaction occurs between the in vivo imaging studies and the biochemical neuroscience and post mortem studies, it has become increasingly evident that RS involves more primarily changes in the cholinergic and glutamatergic system as initial events. Hence, we are redirecting our focus to the study of these systems through functional, structural and metabolic measures using PET/SPECT volumetric MRI and magnetic resonance spectroscopy respectively. In Aim 1, we examine the cholinergic system from 2 aspect. The first is the measurement of viability and cholinergic terminals using SPECT imaging with IBVM. The second is to monitor the effect of acetylcholinesterase drugs on changing intrasynaptic acetylcholine by 11C nicotine imaging. Incorporated with this are monitoring possible changes in regional cerebral blood flow as a function of treatment. Aim 2 will examine the glutamatergic function during ages 2 to 24 years. The hypothesis of hyperexcitability in the glutamate system within the first 2-3 years of age will be tested directly nd compared with RS subjects in later ages using the 11C raclopride technique to measure intrasynaptic dopamine release following ketamine injections. This will be simultaneously studied with MRS measures of glutamate/glutamine. The third aim will be improve on key methods that underlie the continued study of functional and structural measurements. This includes the correction for partial volume effects in PET and SPECT studies which is especially crucial since we will study subjects that vary in ages from 2 through the adult and the volumetrics. The second area is the improvement of anesthetic techniques taking advantage of new anesthesias available for use in in vivo human studies. The fourth aim will be to continue the measures of volumetric changes in specific brain structures. Important recent findings of the changes in the caudate, for example, relative to the rest of the brain provide continuing evidence that relates to the neurochemical measurements. In summary, a functional, structural and metabolic approach obtained in vivo will be used to correlate with the other in vitro and biochemical approaches in clinical approaches in this application to make a concerted effort towards better progress in understanding the pathogenesis of Rett syndrome.